Association of n-acetylcysteine and colistin for use in bacterial infections

ABSTRACT

A synergistic pharmacological association of N-acetylcysteine (NAC) and colistin for use in the treatment of a bacterial infection caused by one or more pathogens selected from  S. maltophilia  and  A. baumannii  strains, in particular a bacterial infection associated with a respiratory tract disease, such as a chronic respiratory tract disease comprising cystic fibrosis (CF), bronchiectasis non CF and chronic obstructive pulmonary disease COPD is described.

This application is a divisional application of U.S. Ser. No. 16/487,227filed Aug. 20, 2019, which is a U.S. National Stage of PCT/EP2018/054611filed on 26 Feb. 2018, which claims priority to and the benefit ofEuropean Patent Application no. 17158241.4, filed on 27 Feb. 2017, thecontents of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to a synergistic pharmacologicalassociation of N-acetylcysteine (hereinafter NAC) and colistin for usein the treatment of bacterial infections caused by one or more pathogensselected from Stenotrophomonas maltophilia (S. maltophilia) andAcinetobacter baumannii (A. baumannii) strains. In particular, thepresent invention relates to a synergistic pharmacological associationof NAC and colistin for use in the treatment of a disease associatedwith bacterial infections caused by one or more pathogens selected fromS. maltophilia and A. baumannii strains, particularly for use in thetreatment of a respiratory tract disease.

BACKGROUND OF THE INVENTION

Colistin, also termed polymixin E, a polymixin antibiotic produced bycertain strains of Bacillus polymixa, consists of a cationic cyclicheptapeptide with a tripeptide side chain acylated at the N terminus bya fatty acid through an a-amide linkage (Reviews of Anti-InfectiveAgents CID 2005; 40: 1033-41).

Two different forms of colistin are available for clinical use: colistinsulfate which is administered orally for bowel decontamination andtopically as a powder for the treatment of bacterial skin infections,and colistimethate sodium (CMS) (also called colistin methanesulfate,pentasodium coli stimethanesulfate, and colistin sulfonyl methate) forparenteral (intravenous, intramuscular, aerosolized andintrathecal/intraventricular) therapy.

Colistin can be therefore administered as a prodrug in form ofcolistimethate sodium, which is readily idrolized to formsulfomethylated derivatives, as well as colistin sulfate, the activeform of the drug.

Colistin has recently gained a crucial role for the treatment of varioustypes of infections (e.g. pneumonia, bacteremia, urinary tractinfections) caused by Gram-negative pathogens expressing a multidrugresistance phenotype (e.g. non-fermenting Gram-negative pathogens, andcarbapenem-resistant enterobacteria).

In patients affected by cystic fibrosis or other chronic respiratorydiseases, it is commonly used to treat infections caused by Pseudomonasaeruginosa (P. aeruginosa) or other multidrug resistant non-fermentingGram-negative pathogens (e.g. Acinetobacter baumannii, Stenotrophomonasmaltophilia).

Colistin represents a last-resort agent for the treatment of infectionscaused by multidrug resistant A. baumannii. In this perspective, greatconcerns arise from the rising trend of resistance to colistin in A.baumannii, which often results in pan-drug resistance phenotypes (PDR),with no available treatment option left.

Treatment of S. maltophilia infections is complicated by intrinsicmultidrug resistance. In case the first line treatment based ontrimethoprim-sulphamethoxazole is not suitable (for patient intoleranceor acquired resistance), colistin is among the second line options, withthe advantage of being also administrable by nebulization. However,increasing colistin resistance rates in S. maltophilia have beendocumented, representing a matter of great concern.

Colistimethate sodium is a combination of the negatively chargedmolecular ion colistin sulphomethate with positive sodium ions.

Colistimethate sodium can be produced by a sulfomethylation reaction ofcolistin with formaldehyde followed by sodium bisulfite, which leads tothe addition of a sulfomethyl group to the primary amines of colistin(Antimicrob. Agents Chemother. April 2003 vol. 47 no. 4 1364-1370).

Colistimethate sodium is commercially available under various brandnames by different manufacturers throughout the world, for example asPromixin™ (Profile Pharma Limited).

According to the present invention the term “colistin” includes colistinand its pharmaceutically acceptable salts and/or prodrugs such ascolistin sulfate, colistimethate sodium, colistin methane sulphate andcolimycin or colomycin, all intended to refer to the bactericidalcyclopeptide antibiotic colystin or precursors thereof such as theprodrug colimycin which is converted to the active drug colistin.

In a particular aspect, for in vitro susceptibility testing in theexperimental part of the present invention the term “colistin” refers tocolistin sulfate (in accordance with the international guidelines forantimicrobial susceptibility testing provided by the Clinical andLaboratory Standards Institute—CLSI and the European Committee onAntimicrobial Susceptibility Testing—EUCAST).

NAC is the acetylated precursor of both the amino acid L-cysteine andreduced glutathione (GSH). Historically it has been used as a mucolyticagent with antioxidant and anti-inflammatory properties, in patients whohave viscid or thickened airway mucus for a range of chronic respiratoryillnesses, including chronic bronchitis, emphysema, COPD andexacerbations, cystic fibrosis, bronchiectasis, as an antidote due toacetaminophen overdose and as a potential treatment of diseasescharacterized by free radical, oxidant damage.

NAC can be typically considered a non-antibiotic drug, to whichantibacterial and antibiofilm properties have been associated in somecases. For example, in 1977, Parry and Neu found that NAC had thecharacteristics to inhibit the growth of both gram-positive andgram-negative bacteria, including Staphylococcus aureus, P. aeruginosa,Klebsiella pneumoniae and Enterobacter cloacae (Journal of ClinicalMicrobiology, January 1977, p. 58-61).

In the literature, controversial results have been reported regardingthe interference of antibiotics and mucolytic agents such as NAC. In1981, Roberts and Cole found that 2%-5% of NAC exhibited antimicrobialactivity against P. aeruginosa and that the effect of the carbenicillinon P. aeruginosa was augmented by low concentrations of NAC (Journal ofInfection Volume 3, Issue 4, December 1981, Pages 353-359).

In 2016, the result of a study carried out by Landini et al. on theeffect of high NAC concentrations on antibiotic activity against acollection of respiratory pathogens, demonstrated that high NACconcentrations do not interfere with the activity of the most commonlyused antibiotics, whereas NAC compromised the activity of carbapenems(Antimicrob. Agents Chemother. December 2016 vol. 60 no. 12 7513-7517).

NAC is commercially available under various brand names by differentmanufacturers throughout the world, for example as Fluimucil™ (ZambonSpa).

S. maltophilia infections are commonly associated with respiratory tractdiseases, in particular chronic respiratory tract diseases; for example,S. maltophilia infections can trigger pulmonary exacerbations of chronicobstructive lung disease (COPD), Cystic Fibrosis (CF) and bronchiectasisnon CF.

S. maltophilia infections occur principally, but not exclusively, indebilitated and immunosuppressed individuals.

S. maltophilia are characterized by a multidrug resistance phenotype andthe ability to form biofilms. In virtue of these features, they areresponsible for chronic lung colonization in individuals with weakenedimmune systems or chronic lung disease, particularly CF andbronchiectasis non CF, which may last several months or years and aredifficult or impossible to be eradicated by current antibiotic treatmentstrategies.

As S. maltophilia, A. baumannii is an important and difficult-to-treatpathogen, which can infect the respiratory tract of patients affected byrespiratory tract diseases, including chronic respiratory tractdiseases. For example, the presence of A. baumannii infections inpatients with pneumonia, in particular, in ventilator-associatedpneumonia (VAP), represents one of the most dreadful complications thatoccur in the critical care setting.

A. baumannii has been also identified in the CF population and in thepopulation with bronchiectasis non CF.

The association of A. baumannii infections with bacteremia, woundinfections, urinary tract infections and meningitis has been alsodescribed. It can be noted that there has been a dramatic increase inthe number of A. baumannii strains with resistance to multipleantibiotic classes and the multidrug-resistant nature of this pathogenrepresents a challenge in the treatment and control of nosocomialinfections, with relatively limited treatment options. For example,nosocomial pneumonia due to multidrug resistant gram-negative bacteriacomprising A. baumannii, is among the most serious complications thatoccur in the intensive care unit (ICU) setting. Mortality, morbidity andhealth care costs are substantially increased by this type of infection.

Despite a significant amount of time and energy has been devoted tostudying effective treatments for dealing successfully with bacterialinfections, especially of the airway and/or lung, caused by a range ofpathogens including emerging pathogens such as S. maltophilia and A.baumannii, there is substantially lack of good therapeutic options andhence there remains a need for better treatments of said bacterialinfections, in particular those associated with chronic lung diseasessuch as, for example, CF, bronchiectasis non CF and COPD.

SUMMARY OF THE INVENTION

Accordingly, the present inventors faced the problem of treating abacterial infection caused by one or more pathogens selected from S.maltophilia and/or A. baumannii.

After a long set of tests and experimentations, the present inventorshave surprisingly found that the association of NAC and colistin has asynergistic antimicrobial effect against a consistent number of S.maltophilia and/or A. baumannii clinical isolates, even resulting in abactericidal effect.

The association of NAC and colistin has also been found to havesynergistic anti-biofilm activity in in vitro biofilm models of S.maltophilia and/or A. baumannii. The anti-biofilm activity of theassociation of NAC and colistin against S. maltophilia and/or A.baumannii biofilms envisages a potential use also for both preventingand eradicating colonization by said pathogens, especially in thehospitalized patients affected by diseases including, for example,chronic bronchitis, emphysema, COPD and exacerbations, CF,bronchiectasis non CF, pneumonia, VAP, bacteremia, wound infections,urinary tract infections and meningitis.

More in detail, the present inventors noticed that with an increase inthe use of colistin to treat infections caused by multidrug resistantGram-negative pathogens, resistance to colistin has been increasinglyreported.

Since colistin represents one of the last therapeutic options to fightGram-negative bacteria, the appearance of colistin-resistantGram-negative pathogens such as S. maltophilia and/or A. baumannii is ofgreat concern.

The present inventors, by concentrating their efforts in identifying aneffective treatment able to restore susceptibility of said pathogens tothe colistin treatment, have unexpectedly found that colistineffectiveness against colistin-resistant Gram-negative pathogens suchas, e.g., A. baumannii and S. maltophilia can be surprisingly restoredby administering colistin together with NAC. In particular, the MIC ofall colistin resistant strains tested lowered at or below thesusceptibility breackpoint in the presence of NAC 8 mg/ml.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Time kill curves. NAC alone (1.6 and 8 mg/ml), colistin alone (2and 8 μg/ml and combination NAC+colistin (1.6 mg/ml+2 μg/ml, 1.6 mg/ml+8μg/ml, 8 mg/ml+2 μg/ml, 8 mg/ml+8 μg/ml). Symbols: -••-: Control; ⋅⋅⋅⋅⋅(dotted line): NAC 1.6 mg/ml;

: NAC 8 mg/ml;

Colistin 2 μg/ml;

Colistin 8 μg/ml; ⋅⋅⋅⋅⋅◯⋅⋅⋅⋅⋅ (dotted line) NAC 1.6 mg/ml+Colistin 2μg/ml; ⋅⋅⋅⋅⋅□⋅⋅⋅⋅⋅ (dotted line): NAC 1.6 mg/ml+Colistin 8 μg/ml;

: NAC 8 mg/ml+Colistin 2 μg/ml;

: NAC 8 mg/ml+Colistin 8 μg/ml.

FIG. 2A). Time kill curves of S. maltophilia isolates with sub MICconcentrations of N-acetylcysteine (NAC 1.6, 3.2 and 8 mg/ml) andcolistin (COL 2, 8 μg/ml) alone and in combination. STX:trimethoprim-sulphametoxazole resistant S. maltophilia strain. Symbols:-×- : Control; —◯ Colistin 2 μg/ml; —□— Colistin 8 μg/ml; —: NAC 1.6mg/ml; ⋅⋅⋅⋅ (dotted line) NAC 3.2 mg/ml; - - - (dashed line) NAC 8mg/ml;

: NAC 1.6 mg/ml+Colistin 2 μg/ml;

: NAC 1.6 mg/ml+Colistin 8 μg/ml;

(dotted line) NAC 3.2 mg/ml+Colistin 2 μg/ml;

(dotted line) NAC 3.2 mg/ml+Colistin 8 μg/ml;

(dashed line): NAC 8 mg/ml+Colistin 2 μg/ml; -

(dashed line): NAC 8 mg/ml+Colistin 8 μg/ml.

FIG. 2B) Time kill curves of S. maltophilia isolates with sub MICconcentrations of N-acetylcysteine (NAC 1.6, 3.2 and 8 mg/ml) andcolistin (COL 2, 8 μg/ml) alone and in combination. S. maltophilia CF:strain from a Cistic Fibrosis patient. Symbols: -×-: Control; —◯—Colistin 2 μg/ml; —□— Colistin 8 μg/ml; —: NAC 1.6 mg/ml; ⋅⋅⋅⋅ (dottedline) NAC 3.2 mg/ml; - - - (dashed line) NAC 8 mg/ml;

: NAC 1.6 mg/ml+Colistin 2 μg/ml;

: NAC 1.6 mg/ml+Colistin 8 μg/ml;

(dotted line) NAC 3.2 mg/ml+Colistin 2 μg/ml;

(dotted line) NAC 3.2 mg/ml+Colistin 8 μg/ml;

(dashed line): NAC 8 mg/ml+Colistin 2 μg/ml;

(dashed line): NAC 8 mg/ml+Colistin 8 μg/ml.

FIG. 3. Antibiofilm activity of NAC, colistin and colistin/NACcombinations (sub-MIC concentrations) tested on a Cystic Fibrosisisolate of S. maltophilia. Biofilms were grown on Calgary Device (MBECassay) for 48 hrs and challenged for 24 hours with NAC alone, Colistinalone at two different concentration: 2 μg/ml and 8 μg/ml and thecombination of the two (NAC 8 mg/ml+Colistin 2 μg/ml and 8 μg/ml).

DETAILED DESCRIPTION OF THE INVENTION

It is therefore a first aspect of the present invention a synergisticpharmacological association of NAC and colistin for use in inhibiting orsuppressing the growth and/or killing a strain of a pathogen selectedfrom S. maltophilia and A. baumannii strains.

It is another aspect of the present invention a synergisticpharmacological association of NAC and colistin for use in the treatmentof a bacterial infection caused by one or more pathogens selected fromS. maltophilia and A. baumannii strains, in particular a bacterialinfection associated with a respiratory tract disease, particularly achronic respiratory tract disease such as e.g., CF, bronchiectasis nonCF and COPD.

In a particular aspect, a bacterial infection is caused by a pathogenselected from S. maltophilia and A. baumannii strains, either expressinga colistin susceptible or resistant phenotype; especially, a pathogenselected from S. maltophilia and A. baumannii strains, expressing acolistin resistant phenotype.

A bacterial infection caused by S. maltophilia strain can be detected,for example, in patients suffering from respiratory tract diseases,particularly chronic respiratory tract diseases such as, for example, inpatients suffering from COPD, CF or bronchiectasis non CF, especiallywhen these patients have weakened immune systems.

A bacterial infection caused by A. baumannii strain can be detected, forexample, in patients suffering from pneumonia, VAP, bacteremia; woundinfections, urinary tract infections, CF and bronchiectasis non CF,especially when these patients are hospitalized patients.

In a more particular aspect of the present invention, patients sufferingof a bacterial infection caused by a pathogen selected from S.maltophilia and A. baumannii strains as described above areimmunocompromised and/or hospitalized patients.

A bacterial infection caused by a pathogen selected from S. maltophiliaand A. baumannii strains according to the present invention may be abiofilm-associated infection.

The above aspects are based on the observation that there is asynergistic interaction between NAC and colistin, which provides asignificant antibacterial effect.

According to the present invention the terms “synergistic” and“synergistically” as applied to the effect of NAC and colistin used inassociation (whether simultaneously or sequentially) refer to a greaterantibacterial effect obtained when the above-identified bacteria aretreated by either NAC or colistin alone. In some embodiments, the effectof NAC and colistin used in association (whether simultaneously orsequentially) is greater than the simple addition of the effects of eachagent administered alone, i.e. there is an effect which surpassesexpectations based on additive effects.

It is a further aspect of the present invention a synergisticpharmacological association of NAC and colistin for use in the treatmentof a bacterial infection caused by one or more pathogens selected fromcolistin-resistant S. maltophilia and colistin-resistant A. baumanniistrains, wherein NAC is able to restore susceptibility of said pathogensto colistin.

It is a further aspect of the present invention a pharmacologicalassociation of NAC and colistin for use in the treatment of a bacterialinfection caused by one or more pathogens selected fromcolistin-susceptible S. maltophilia and colistin-susceptible A.baumannii strains, wherein NAC is able to prevent in vivo emergence ofcolistin resistance, during colistin treatment regimens.

NAC and colistin of the pharmacological association according to thepresent invention may be administered in either order, separately orconcurrently, with overlapping or non-overlapping periods ofadministration, via the same or different modes of administration.

For example, NAC and colistin can be concurrently administered in asingle dosage form or alternatively NAC and colistin can be administeredin separate dosage forms in either order, concomitantly or sequentially,with overlapping or non-overlapping periods of administration, via thesame of different modes of administration.

The concurrent or separate administration of the pharmacologicalassociation of NAC and colistin has the effect of in inhibiting orsuppressing the growth and/or killing a strain of a pathogen selectedfrom S. maltophilia and A. baumannii strains, said effect beingunexpectedly and surprisingly greater than what is seen when bacteriaare contacted by either NAC or colistin alone.

In detail, the present inventors noticed that with an increase in theuse of colistin to treat infections caused by multidrug resistantGram-negative pathogens, resistance to colistin has been increasinglyreported.

Since colistin represents one of the last antibiotic resorts to fightGram-negative bacteria, the appearance of colistin-resistantGram-negative pathogens is of great concern.

The present inventors strongly perceived the great need of an effectivetreatment against colistin-resistant Gram-negative pathogens such as,e.g., A. baumannii and S. maltophilia, able to restore susceptibility ofsaid pathogens to the antibiotic treatment, or to prevent in vivoemergence of colistin resistance during colistin treatment regimens.

The present inventors have found that colistin effectiveness againstcolistin-resistant

Gram-negative pathogens such as, e.g., A. baumannii and S. maltophiliacan be surprisingly restored by the administration of the association ofNAC with colistin.

In particular, colistin susceptibility can be restored by NACconcentrations possibly achievable by topical administration. Inparticular, the MIC of all colistin resistant strains tested lowered ator below the susceptibility breakpoint in the presence of NAC 8 mg/ml.

The association of NAC and colistin may be administered to the patientin the form of one or more pharmaceutical formulations.

The pharmaceutical formulations of the present invention comprise NACand/or colistin together with a carrier suitable for pharmaceutical use,consisting of one or more excipients. For example, the pharmaceuticalformulations of the invention may comprise both NAC and colistintogether with a carrier suitable for pharmaceutical use, consisting ofone or more excipients, i.e. pharmaceutical formulations for concurrentadministration of both NAC and colistin; or the pharmaceuticalformulations of the invention may comprise NAC together with a carriersuitable for pharmaceutical use, consisting of one or more excipients;or the pharmaceutical formulations of the invention may comprisecolistin together with a carrier suitable for pharmaceutical use,consisting of one or more excipients, i.e. separate pharmaceuticalformulations for sequential or concomitant administration of NAC andcolistin.

According to the present invention, the term “excipient” comprises anyinert substance added to a pharmaceutical composition to furtherfacilitate administration of an active ingredient.

According to the present invention, the term “carrier” comprises anysubstance suitable as a vehicle for delivering NAC and/or colistin to asuitable in vivo or in vitro site.

Pharmaceutically acceptable excipients are those compounds well known toa skilled person in the art that can be used to produce formulationscomprising NAC and/or colistin that are suitable to be administered to asubject. Acceptable methods for preparing the pharmaceuticalformulations according to the invention are well known to a personskilled in the art.

It is a further object of the present invention a method of treating abacterial infection caused by a pathogen selected from S. maltophiliaand A. baumannii strains in a subject in need thereof, which comprisesadministering an association comprising NAC and colistin, wherein theassociation has a synergistic antibacterial effect.

It is a further object of the present invention a method of treating abacterial infection caused by a pathogen selected from S. maltophiliaand A. baumannii strains in a subject in need thereof, which comprisesconcurrently administering an association comprising NAC and colistin,wherein the association has a synergistic antibacterial effect.

It is a further another object of the present invention a method oftreating a bacterial infection caused by a pathogen selected from S.maltophilia and A. baumannii strains in a subject in need thereof, whichcomprises separately administering an association comprising NAC andcolistin, wherein the association has a synergistic antibacterialeffect.

According to the present invention, the terms “individual” “subject” and“patient” are used interchangeably to refer to a member of mammalianspecie, preferably a human, which is afflicted with a particulardisease, disorder or condition.

According to the present invention the term “antibacterial” meansreducing the harmful effects of bacteria by inhibiting, suppressing thegrowth and/or killing them. According to the present invention the term“antibacterial agents” includes NAC and colistin.

According to the present invention the term “bactericidal” means havinga destructive killing action upon bacteria.

According to the present invention, “bacterial infection” refers to anysituation in which the presence of a microbial population(s) is damagingto a host mammal. Thus, an individual is “suffering” from a microbialinfection when excessive numbers of a microbial population are presentin or on an individual's body, or when the effects of the presence of amicrobial population(s) is damaging the cells or other tissue of anindividual. In particular, “bacterial infection” refers to an infectioncaused by a strain of bacteria for which the use of a synergisticpharmacological association of NAC and colistin disclosed herein isappropriate.

According to the present invention, the terms “treat”, “treating” and“treatment” refer to a diminution, a decrease, a limitation, or amitigation of the degree, intensity, extent of a bacterial infection orits related disease conditions and symptoms caused by a strain of S.maltophilia and/or A. baumanii, that is achieved by a reduction,inhibition or suppression of growth, replication, and/or propagation, ordeath or destruction of said bacteria, on or in the subject.

According to the present invention, the term “pharmacologicalassociation” refers to either a fixed combination of NAC and colistin inone unit dosage form, a non-fixed combination or a kit of parts for thecombined administration where NAC and colistin, as defined above, may beadministered simultaneously, independently at the same time orseparately within time intervals that allow the combination partners toshow a synergistic effect.

Any suitable route of administration may be used for the compositions ofthe present invention, including oral, parenteral (subcutaneous,intramuscular or intravenous) and inhalation route.

According to the present invention, the terms “oral” or “orally” referto the introduction into the body by mouth whereby absorption occurs inone or more of the following areas of the body: the mouth, stomach,small intestine, and the small blood vessels of the oral mucosa.

Non-limiting examples of pharmaceutical formulations according to thepresent invention for oral administration include, for example, tablets,coated tablets, granulates, pills, capsules, liquids, gels, syrups,suspensions, and the like, for oral ingestion by an individual. Suitablecarriers for oral administration are well known in the art.

For parenteral administration, pharmaceutical formulations according tothe invention may be formulated for example in aqueous solutions such asin physiologically compatible buffers or physiological salt buffer.Formulations for injection may be presented in unit dosage forms, forexample, in ampoules, or in multi-dose containers with, optionally, anadded preservative.

For administration by inhalation route, pharmaceutical formulationsaccording to the invention may be formulated in solutions, suspensionsand dry powder and delivered by using conventional means, so thatoptimal quantities of a suitable range of particle sizes are provided tothe patient.

The pharmaceutical formulations according to the invention may bepreferably administered to the respiratory tract. Thus, the presentinvention also provides aerosol pharmaceutical formulations comprisingNAC and/or colistin.

Pharmaceutical compositions of the present invention may be manufacturedin conventional manners, following processes well known in the art.

The amount of NAC and colistin for use according to the presentinvention may vary depending on the administration route, the selectedkind of composition, the individual characteristics of the patient, theduration of the treatment and the nature of concurrent therapies.

For example, the synergistic effective amount of the pharmacologicalassociation of NAC and colistin can produce a diminution, a decrease, alimitation, or a mitigation of the degree, intensity, extent of abacterial infection or its related symptoms caused by a strain of A.baumannii and/or S. maltophilia.

According to one embodiment, the amount of NAC and colistin sufficientto have a synergistic effect on a bacterial infection caused by a strainof A. baumannii and/or S. maltophilia may vary, for example, in view ofthe physical characteristics of the patient, the severity of thesubject's symptoms, the form of the infection, the identity of thebacteria, the formulations and the means used for administering thedrug. The specific dose for a given subject is usually set by thejudgement of the attending physician.

However, as an example, an effective amount of colistin may be betweenabout 0.075 million units and 12 million units (i.e. between about 6 mgand 960 mg), preferably between about 0.5 million units and 12 millionunits (i.e. between about 40 mg and 960 mg); the effective amount of NACmay vary between 100 and 5800 mg, preferably between 100 and 4600 mg, tobe administered in a single dose or in more repeated doses.

Depending of the means of administration a dose may be administered allat once or slowly over a period of time, such as with an i.v. or byinhalation administration.

According to the present invention, the term “dose”, “unit dose”,“dosage”, “effective dose” and related terms refer to physicallydiscrete units that contain a predetermined quantity of activeingredient calculated to produce a desired therapeutic effect. A singledose is thus a predetermined quantity of colistin or NAC that isadministered to a patient.

The exact composition, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition.Treatment should be continued for as long as required to receive thebenefit of the invention.

A determination of a synergistic interaction between NAC and colistinmay be based on the results obtained from the assays described herebelow.

In vitro antimicrobial synergism between NAC and colistin was determinedfollowing EUCAST guidelines. In particular, the total fractionalinhibitory concentration (ΣFIC) was calculated as follows: ΣFIC equalsFIC of agent A plus FIC of agent B, where the FIC of agent A or B is theminimum inhibitory concentration (MIC) of agent A or B in the presenceof the other divided by the MIC of agent A or B alone. Synergism wasdefined as a ΣFIC value of <0.5.

EXAMPLE 1

Activity of NAC in Association with Colistin Against A. baumannii

Seven colistin resistant A. baumannii clinical isolates were included inthe study (colistin MIC range 16-256 μg/ml, MIC50=64 MIC90=128 μg/ml).Classic checkerboard assays were used to investigate potential synergismbetween colistin (range 0.25-256 μg/ml) and NAC (range 0.5-32 mg/ml).Fraction inhibitory concentration indices (FICIs) were calculated, andsynergism was defined as FICI values <0.5. Synergy between colistin andNAC was also confirmed by time-kill assays performed with one isolate,using two different concentrations of colistin (2 and 8 μg/ml,representing concentrations achievable in serum and epithelial liningfluid—ELF, respectively) and NAC (1.6 and 8 mg/ml, likely achievable inELF by topical administration).

The MIC of NAC was 32 mg/ml for two isolates, and >32 mg/ml for theremaining ones. Synergism between colistin and NAC was observed incheckerboard assays with all tested isolates. In particular, arestoration of colistin susceptibility (i.e. MIC≤2 μg/ml) was observedwith 100% and 52% of tested strains in the presence of NAC 4 mg/ml andNAC 2 mg/ml, respectively. Time-kill curves confirmed the synergyobserved by checkerboard assays, demonstrating a bactericidal effect ofcolistin/NAC combinations at sub-MIC concentrations (FIG. 1 shows thecurves obtained with isolate N50, with NAC MIC=>32 mg/ml and ColistinMIC=16 μg/ml).

In order to further investigate the synergism of colistin/NACcombinations against colistin-resistant A. baumannii strains, time-killassays were also performed according to CLSI guidelines with twoselected strains (i.e. Z165 and Z167, Col^(R)). Two colistinconcentrations (i.e. 2 and 8 μg/ml), and three NAC concentrations (i.e.1.6, 3.2 and 8 mg/ml) were tested alone and in combination withcolistin, with determination of viable cells performed after 0, 2, 4, 6,8, 24 and 48 hours of exposure (detection limit 25 CFU/ml). Data wereobtained in two independent experiments, with two replicates percondition per experiment and show a dose-dependent potentiation ofcolistin activity by NAC. In particular, a complete eradication of thestarting inocula was achieved with combinations including NAC 8 mg/ml(i.e. absence of regrowth after 48 hours of incubation).

A biofilm eradication test on the two isolates was also carried out asfollows: biofilms were grown in the NUNC TSP lid system (MBEC assay),for seven days in daily refreshed CAIVIHB (static conditions). Preformedbiofilms were then exposed to two colistin concentrations (i.e. 2 and 8μg/ml), and three NAC concentrations (i.e. 1.6, 3.2 and 8 mg/ml), aloneand in combination, for 24 hours. After exposure, biofilms weredisrupted by sonication and mean viable cell count per peg (log CFU/peg)was determined (detection limit 1.3 log CFU/peg). Data were obtained inat least two independent experiments, with at least six replicates percondition per experiment.

Results showed a remarkable antibiofilm synergistic activity ofcombinations including colistin 8 μg/ml (i.e. representing 0.25× and0.06×MIC for Z165 and Z167, respectively), with a marked reduction ofviable biofilm cells also observed in the presence of the lower NACconcentration tested (i.e. 1.6 mg/ml).

EXAMPLE 2

Activity of NAC in Association with Colistin Against S. maltophilia

Twenty S. maltophilia clinical isolates were tested, including also fourisolates from patients affected by cystic fibrosis (CF). Two isolatesshowed resistance to trimethoprim-sulphamethoxazole. Synergism betweencolistin (range 0.25-256 μg/ml) and NAC (range 0.5-32 mg/ml) wasinvestigated by classic checkerboard assays. Fraction inhibitoryconcentration indices (FICIs) were calculated, and synergism was definedas FICI values <0.5. Time-kill assays were performed with two clinicalisolates (including one strain resistant totrimethoprim-sulphamethoxazole, and one from CF). For this purpose, twodifferent concentrations of colistin (2 and 8 μg/ml, representingconcentrations achievable in serum and epithelial lining fluid—ELF,respectively) and NAC (1.6 and 8 mg/ml, likely achievable in ELF bytopical administration) were tested alone and in combination.

Colistin MICs ranged from 0.5 μg/ml to 128 μg/ml (MIC50, 16 μg/ml;MIC90, 64 μg/ml). The MIC of NAC was 16 mg/ml for nine isolates, >32mg/ml for one isolate, and 32 mg/ml for the remaining ones. Synergismbetween colistin and NAC was observed in checkerboard assays with alltested isolates. In particular, NAC 8 mg/ml and 2 mg/ml lowered colistinMIC to ≤2 μg/ml (the susceptibility breakpoint for A. baumannii and P.aeruginosa) for 100% and 47% of isolates with colistin MIC>2 μg/ml(n=17), respectively. Time-kill curves confirmed the synergy observed bycheckerboard assays, demonstrating a bactericidal effect of colistin/NACcombinations at sub-MIC concentrations (FIG. 2A and FIG. 2B).

The antibiofilm activity of colistin/NAC combinations was investigatedusing the MBEC assay, the reference procedure for biofilm susceptibilitytesting (. Mature S. maltophilia (48-hours old) were challenged for 24hours with NAC and colistin, either alone or in combination. Viable cellcount of challenged biofilms was compared to controls in order to assessthe antibiofilm activity of the tested drugs and drugs/combinations.

Sub-MIC colistin/NAC combinations were also found to exert a relevantantibiofilm activity against mature S. maltophilia biofilms. Inparticular, NAC 8 mg/ml plus colistin 2 μg/ml accounted for a reductionof more than 3 log colony forming units (CFU)/peg compared to controls(FIG. 3).

The results show that MIC of all colistin resistant strains tested (i.e.S. maltophilia, n=17; A. baumannii, n=7) lowered at or below thesusceptibility breakpoint in the presence of NAC 8 mg/ml.

1. A method of restoring susceptibility to antibiotic treatment incolistin-resistant S. maltophilia and colistin-resistant A. baumanniistrain during or after bacterial infection caused by said one or morepathogens in patients in need thereof, said method comprisingadministering to said patients a pharmaceutical effective amount of asynergistic pharmacological association consisting of N-acetylcysteine(NAC), colistin and a carrier.
 2. The method according to claim 1,wherein the infection is a biofilm-associated infection.
 3. The methodaccording to claim 1, wherein the infection is detected in patients witha respiratory tract disease.
 4. The method according to claim 3, whereinthe respiratory tract disease is a chronic respiratory tract diseasecomprising Cystic Fibrosis (CF), bronchiectasis non CF and chronicobstructive pulmonary disease (COPD).
 5. The method according to claim1, wherein said bacterial infection caused by S. maltophilia strain isdetected in patients suffering from a respiratory tract disease.
 6. Themethod according to claim 1, wherein the bacterial infection caused byA. baumannii strain is detected in patients suffering from a respiratorytract disease.
 7. The method according to claim 1, wherein the patientsare immunocompromized and/or hospitalized patients.
 8. The methodaccording to claim 1, wherein NAC and colistin are administered ineither order, separately or concurrently, with overlapping ornon-overlapping periods of administration.
 9. The method according toclaim 8, wherein NAC and colistin are administered via the same ordifferent administration routes.
 10. The method according to claim 1,wherein NAC and colistin are concurrently administered in a singledosage form.
 11. The method according to claim 1, wherein NAC andcolistin are administered in separate dosage forms in either order,concomitantly or sequentially, with overlapping or non-overlappingperiods of administration.
 12. A method of disrupting biofilm formationin colistin-resistant S. maltophilia and colistin-resistant A. baumanniistrains during bacterial infection caused by said one or more pathogensin patients in need thereof, said method comprising: administering tosaid patients a pharmaceutical composition consisting of apharmaceutical effective amount of NAC together with a carrier.